Technical Abstract:
Commercial catfish production accounts for 85-90% of the total fin fish aquaculture production in the United States, with almost 300,000 tonnes produced in 2003. Significant losses caused by the Gram negative bacterial pathogen Edwardsiella ictaluri were reported on over 60% of all farms in operation. Although there is substantial descriptive data relative to invasion, spread and persistence of E. ictaluri in channel catfish, little is known about the virulence factors involved in the process. Chondroitinase and lipopolysaccharide (LPS) have been implicated in
the pathogenesis of E. ictaluri, and several reports allude to intracellular replication and survival in catfish macrophages and neutrophils. We have recently reported entry, survival, and replication of E. ictaluri in vitro in head-kidney-derived macrophages (HKDM) and there are also reports of invasion into fish cell lines, but there are no reports regarding the mechanism of this activity.
Signature-tagged mutagenesis (STM) is a mutagenesis system involving mini-transposons that carry unique DNA tags, enabling the identification of individual mutants in a mixture of mutants. Briefly, a mixture of tagged mutants are used to establish an infection and the mutants that are present initially are later compared to the mutants that remain at the death of the host. Those lost during the infection process are presumed to be attenuated as a result of an insertion of the transposon into a gene that is required for survival in the host. Because the tagged transposons carry an antibiotic resistance marker, the transposon and the region flanking the transposon can be sub-cloned from a restriction digest of genomic DNA using
antibiotic selection. The sub-cloned region carrying the insertion can be identified using the tag as a primer to sequence the DNA flanking the transposon and comparing the sequence to known bacterial DNA databases.
Because very little is known about the factors involved in E. ictaluri pathogenesis, STM was used to screen 1071 mutants in a waterborne infection model, resulting in the identification of 52 that were unable to survive in the catfish host. Nine mutations were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in LPS biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. Using the sequence of the TTSS associated genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135 bp pathogenicity island
encoding 33 genes of a TTSS with similarity to the Salmonella pathogenicity island-2 encoded TTSS and high similarity to the TTSS of E. tarda. A TTSS mutant retained its ability to invade catfish cells, but was defective in intracellular replication. The mutant also invaded catfish tissues in equal numbers to the wild-type E. ictaluri following infection by immersion, but replicated poorly and was slowly cleared from the tissues. Taken in conjunction with data indicating that E. ictaluri can survive in conditions down to pH 3 and that E. ictaluri encodes
an acid-inducible urease operon, a model for E. ictaluri survival and replication in catfish macrophages is proposed. Following uptake into the cell, E. ictaluri survives initial acidification of the phagocytic vacuole and the E. ictaluri urease operon is activated, resulting in the production of ammonia. The E. ictaluri phagosome returns to a more neutral pH, which fails to activate acid-inducible lytic enzymes introduced by the lysosome. The E. ictaluri TTSS translocates effector molecules across the vacuolar membrane into the cytoplasm, where, based on homology to similar effector molecules in Salmonella, they conscript the cytoskeletal apparatus of the macrophage and induce the formation of a “spacious phagosome” to
accommodate E. ictaluri replication. Other roles for the E. ictaluri effector molecules remain to be determined.